Preparation of DNA from Cryptosporidium parvum Oocysts for PCR Detection / 中国寄生虫学与寄生虫病杂志

ABSTRACT

Objective To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR. Methods After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex-100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C.parvum gene (L16996), a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex-100 method was also tested by PCR. Results One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst. Conclusion The three kinds of extraction can all be served as templates for PCR detection of C.parvum oocysts, while Chelex-100 method is simpler, quicker and more reliable for DNA extraction of the parasite.